This is an application for the first competitive renewal of RO1 AI45463 "Modified envelope glycoproteins for HIV- 1 vaccine". Our overall goals are to gain additional knowledge of what forms of the HIV- 1 envelope glycoproteins might make effective immunogens for the induction of broadly neutralizing antibodies, and to learn about the properties of these proteins. Towards these goals, we propose three Specific Aims. 1: To study and compare the properties of cleaved, stabilized and uncleaved forms of oligomeric I-IIV-1 envelope glycoproteins. We seek to study purified monomeric, dimeric and trimeric forms of both cleaved and uncleaved HIV-1 JR-FL gpl40 proteins. An emphasis will be to better define the differences between cleaved and uncleaved trimers. We will t study the receptor-binding properties of the different Env forms; their reactivities with neutralizing and non-neutralizing MAbs; their stabilities to heat and detergent, and the pathways by which they dissociate under these conditions; their performance in ultra-centrifugation assays of molecular size; their appearance in electron micrographs both alone and as antibody complexes. 2:To evaluate the immunological responses elicited by cleaved, stabilized and uncleaved forms of oligomeric HIV-1 envelope glycoproteins in small animals. We will conduct immunogenicity studies in rabbits and guinea-pigs to determine whether cleaved, uncleaved and further modified forms of the HIV-1 Env complex can elicit neutralizing antibodies. These studies will involve immunization of the animals in a DNA-prime, protein-boost format, using in the priming phase both membrane-bound and soluble forms of HIV- 1 Env as a comparison. We will also evaluate the immunogenicity of Env complexes captured onto nanometer-sized beads to form particulate antigens. 3: To design, express and evaluate antigenic variants of the I-IIV-1 Env complex which contain additional modifications that may improve their immunogenicity. Unmodified forms of the HIV-1 Env complex may fail to induce broadly neutralizing antibodies, however they are presented. In anticipation of this outcome, we will make oligomeric forms of Env that contain additional modifications designed to enhance their immunogenicity. We will remove specific glycans and/or variable loops, create complexes containing CD4-mimetics, and attempt to induce Env configurations that resemble those that exist subsequent to CD4 binding.